The growth of agricultural crops is generally limited by the availability of nitrogen, and at least 50% of global requirement is met by the application of synthetic fertilisers in the form of ammonia, nitrate or urea. However, there is a growing need to exploit one of the most important natural sources of nitrogen for agriculture, namely biological nitrogen fixation.
The primary source of biological nitrogen fixation are Rhizobium or Rhizobia spp and the actinobacterium Frankia spp, which are a small group of prokaryotes that produce nitrogenases, and form endosymbiotic associations with plants conferring the ability to fix nitrogen. Although many plants can associate with nitrogen-fixing bacteria, only a few plants, all members of the Rosid I Clade, form endosymbiotic associations with Rhizobia spp and Frankia spp., which are unique in that most of the nitrogen is transferred to and assimilated by the host plant. The Leguminosae plant family, which includes soybean, bean, pea, peanut, chickpea, cowpea, lentil, pigeonpea, alfalfa and clover, are the most agronomically important members of this small group of nitrogen-fixing plants. Biological nitrogen fixation via the endosymbiotic association reduces the need for expensive nitrogen fertilizers in legume crops and is an important feature of sustainable agriculture. Legumes can also utilize nitrogen available in the soil, such that when levels of soil nitrate are high, nodule formation is suppressed and the plant shifts from nitrogen metabolism to growth on nitrate (Wopereis et al., 2000).
Rhizobium-legume symbiosis involves the interaction of a set of plant and bacterial genes in a complex process leading to the initiation and development of root nodules. Organogenesis of nodules is triggered by the rhizobial microsymbiont, but the legume host plant encodes the developmental program responsible for building the nodule tissues and for regulating the process. Lipo-chitin-oligosaccharides (Nod-factors) synthesized and secreted by Rhizobia are major signal molecules that trigger this process. The major Nod-factor secreted by the Mesorhizobium loti microsymbiont of Lotus is a pentameric N-acetylglucosamine carrying a cis-vaccenic acid and a carbamoyl group at the non-reducing terminal residue together with a 4-O-acetylfucose at the reducing terminal residue. Perception of Nod-factor in Lotus is mediated by NFR1 and NFR5 receptor kinases (Radutoiu et al., 2003 Nature 425: 585-592; Madsen et al., 2003 Nature 425: 637-640), that together with an LRR receptor-kinase gene, SymRK, communicate with a common signal transduction pathway, shared with mycorrhizal symbiosis (Oldroyd and Downie, 2004 Mol. Cell Biology 5: 566-576). This common pathway is encoded by seven genes, SymRK, Castor, Pollux, Nup133, Sym15, Sym6 and Sym24. Analysis of mutants has shown that NFR1/NFR5 receptor(s), SymRK encoded LRR protein kinase, CASTOR/POLLUX cation channel(s) and nucleoporin133 are required for the induction of calcium spiking, one of the earliest physiological responses detectable in root hairs exposed to purified Nod-factor.
To establish symbiosis, Rhizobia gain access to single plant cells where they are installed in symbiosomes surrounded by a peribacteroid membrane. In Lotus, infection occurs via an infection thread that takes the bacteria through root hairs into the root cortex and distributes them to cells, which become infected symbiosome containing nitrogen-fixing cells. In response to attached bacteria, root hairs deform and curl, setting up a pocket that provides a site for infection thread initiation (Geurts et al., 2005 Curr. Opinion Plant Biol., 8: 346-352). Infection threads are plant-derived structures originating from plasma membrane invagination, accompanied by external deposition of cell wall material. In advance of the inward progressing intracellular thread, root cortical cells dedifferentiate and re-enter the cell cycle to initiate the nodule primordium. Later in the process, pattern formation and cell differentiation specify tissue and cell types including the infected cells that endocytose Rhizobia. In the mature functional nodule, peripheral vascular bundles are connected to the root vasculature and the main tissues/cell types can be distinguished (Pawlowski and Bisseling, 1996, Plant Cell 8: 1899-1913).
Analysis of a group of nodulation mutants, including some that fail to show calcium oscillations in response to Nod-factor signals, has revealed that in addition to the lack of nodulation, these mutants are unable to form endosymbioses with arbuscular mycorrhizal fungi. This implies that a common symbiotic signal transduction pathway is shared by two types of endosymbiotic relationships, namely root nodule symbiosis, which is largely restricted to the legume family, and arbuscular mycorrhizal symbiosis, which is common to the majority of land plant species. This suggests that there may be a few key genes which dispose legumes to engage in nodulation, and which are missing from crop plants such as cereals. The identification of these key genes, which encode functions which are indispensable for establishing a nitrogen fixing system in legumes, and their transfer and expression in non-nodulating plants, has long been a goal of molecular plant breeders. This could have a significant agronomic impact on the cultivation of cereals such as rice, where production of two harvests a year may require fertilisation with up to 400 kg nitrogen per hectare.
In summary, there is a need to transfer the nodule formation capability and nitrogen fixation properties of legume crops into non-nodulating crops in order to meet the nutritional needs of a growing global population, while minimising the future use of nitrogen fertilisers and their associated negative environmental impact.